Success of hybrid poplar plantations will rely on the efficient management of nutrients and weeds. Relatively little is known about the root uptake characteristics of hybrid poplar and weeds, their belowground interactions and particularly, the quantitative understanding of nutrient uptake using mechanistic models under weed-competing conditions. Therefore, the objectives of this study were to investigate the effects of dandelion and quackgrass on the growth of hybrid poplar, to establish their root uptake characteristics and to quantify their nutrient uptake using the soil supply and nutrient demand (SSAND) model. In a pot study, hybrid poplar stem height, root collar diameter, shoot and root biomass, root length, and N, P and K uptake significantly decreased in the presence of dandelion and quackgrass weeds. Similar weed competition effects on growth of hybrid poplar were also observed in the field at the Pasture and Alfalfa sites where hybrid poplar was grown with and without weeds for 50, 79 and 100 days. In a hydroponic experiment, Imax values for NH4-N, NO3-N, P and K varied significantly among hybrid poplar seedlings and dandelion and quackgrass weed species and was greatest for dandelion followed by hybrid poplar and then quackgrass. The Km values were lowest for quackgrass compared to the other plant species for all of the nutrients. Simulation results from the SSAND model for the pot study showed that N uptake was underpredicted in hybrid poplar by 58 to 73%, depending upon soil type and weed treatment. Incorporation of N mineralization as a model input improve the hybrid poplar N uptake predictions by 24 and 67% in the Pasture and Alfalfa soil, respectively, when grown without weeds. SSAND model underestimated P uptake by 84-89% and overestimated K uptake by 28 to 59% for hybrid poplar depending upon the soil type and weed treatment. In the field, N uptake by hybrid poplar was in close agreement to measured N uptake in the control treatment. N uptake was greatly underestimated for both hybrid poplar and weeds in the weed treatment. Including changing water content greatly improves the N uptake by hybrid poplar and weeds in weed treatments. Results from this study suggest weed control is an essential practice to establish successful hybrid poplar plantations. Also, SSAND model can be an effective tool for predicting the nutrient uptake under two plant species competing environment if all the processes of nutrient supply are adequately described in the model.

Vitamin K is a fat soluble compound which is synthesized by the gut microbiota and produced in many tissues within the body. Considering its role in the liver as a cofactor for gamma carboxylase enzymes, treatment of dark circles and pigments under the eye among others. It is clear that is some circumstances vitamin K has to cross biological barriers, particularly, when the vitamin is produced by microbiota in the intestine or applied topically on skin. Thus it is important to develop methods that allow studies of vitamin K permeability through the skin including its participation in redox reactions and transdermal permeability. Taking into account that transdermal permeability is strongly limited for high molecular weight compounds, i.e., compounds with higher than 500Da, the study was conducted with vitamin K of  lower molecular weight. Specifically vitamin K4 model, i.e., 1,4-dihydroxy-2 naphthoic acid, with molecular weight of 204g/mol. Vitamin K4 is suitable for this kind of study , because it can work as reducing (antioxidant) compound as well as has relatively beneficial physicochemical characteristics for transdermal permeability. Permeability studies were conducted with skin covered oxygen electrode and franz diffusion cell. Data from measurements were analyzed to estimate diffusion coefficients, apparent Michaelis-Menten constants and flux of a vitamin K4 model whilst contribution of different permeability pathways was determined theoretically.

Relative to other metals, little is known about lead toxicity in fish. The use of predictive models such as the biotic ligand model (BLM) has been limited, a situation that is at least partially due to the lack of understanding of lead's acute toxic mechanism and characterization of key binding sites involved in this toxicity. Using the rainbow trout as a model species, the acute toxic mechanism for lead was found to be ionoregulatory disruption. While having no apparent respiratory or acid/base effects, Pb exposure resulted in significant ionoregulatory impacts that affected Ca2+ homeostasis, as well as Na+ and Cl- balance. Active Ca2+ uptake by the gills obeyed typical Michaelis-Menten kinetics, and Pb interacted in a competitive fashion with the uptake process. Exposure to increasing waterborne Pb concentrations resulted in significant increases in Km value while Jmax showed little or no change. A slower, non-competitive interaction occurred after prolonged Pb-exposure, evidenced by a significant reduction of high-affinity Ca2+ -ATPase activity that correlated well with branchial Pb accumulation. Conversely, calcium had a protective effect against branchial Pb accumulation, this relationship being predominately competitive in nature. Voltage-independent calcium channel blockers La3+, Cd, and Zn significantly reduced gill Pb burden while the voltage-dependent, L-type calcium channel blockers, nifedipine and verapamil, did not, suggesting Pb enters fish by a similar mechanism to that of Ca2+. Stimulated stanniocalcin release by CaCl2 injection also significantly reduced branchial Pb accumulation. Based on the evidence presented in this thesis, it is apparent that acute Pb toxicity occurs by ionoregulatory disruption. It is likely that Pb shares a similar uptake pathway as that for Ca2+ and that resulting accumulation results in disruption of Ca2+ influx as well as Na+ and Cl- balance. This study has provided data essential to the characterization of key binding sites involved in Pb toxicity, and ultimately, validates the development and application of predictive models such as the BLM. / Thesis / Master of Science (MSc)

The flavonols are a varied and abundant sub-class of flavonoids that are associated with a number of essential physiological functions in plants and pharmacological activities in animals. The 2-oxoglutarate-dependant dioxygenases(2-ODDs), flavonol synthase (FLS) and flavanone 3-hydroxylase (F3H), are essential for flavonol synthesis. The primary goal of this study has been to gain a deeper understanding of the biochemistry of these enzymes in Arabidopsis. To accomplish this goal, an activity assay employing recombinant protein expression and HPLC as a detection system was developed for F3H and adapted for use with FLS. The assay was employed to establish the biochemical parameters of F3H from Arabidopsis, and to further characterize the F3H mutant allele, <i>tt6</i>(87). Enzymatic activity was demonstrated for F3H enzymes from <i>Ipomoea alba</i> (moonflower), <i>Ipomoea purpurea</i> (common morning glory), <i>Citrus sinensis</i> (sweet orange), and <i>Malus X domestica</i> (newton apple), each of which had previously been identified solely based on sequence homology. Arabidopsis contains six genes with high similarity to <i>FLS</i> from other plant species; however, all other central flavonoid pathway enzymes in Arabidopsis are encoded by single genes. The hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation was tested. Sequence analysis revealed that <i>AtFLS2, 4</i> and <i>6</i> contain premature stop codons that eliminate residues essential for enzyme activity. AtFLS1 was found to have a strong preference for dihydrokaempferol as a substrate. However, no enzyme activity was observed for AtFLS3 or AtFLS5 with a number of different substrates under a variety of reaction conditions. To identify structural elements that may contribute to the observed differences in biochemical activity, homology models for each of the isoforms were generated utilizing Arabidopsis anthocyanin synthase (ANS) as a template. A domain at the N-terminus of AtFLS1 that is missing in the other isozymes was insufficient to convey activity to an AtFLS1/5 chimera. These findings suggest a single catalytically-active form of FLS exists in Arabidopsis. The possibility that the apparently expressed but non-catalytic proteins, AtFLS2, 3, and 5, serve noncatalytic roles in flavonol production were explored by yeast 2-hybrid analysis. / Ph. D.

Es sind Begriffe aus dem kulturellen Erbe, die heute weitgehend unbekannt sind und mit der Industrialisierung aus dem Wortschatz verschwanden. Jeweils Ostern endete das Schuljahr. Besagte Ostermädchen und -jungen waren Schulabgänger der Volksschule, die mit 14 Jahren die gesetzlich vorgeschriebene achtjährige Volksschulbildung abgeschlossen hatten und mit dem Ende der Schulzeit zu Ostern als Ungelernte in billige Dienste bei Bauern oder als Hilfskraft bei kleineren Handwerkern gehen mussten, um ihre Familien zu unterstützen.

Els hidrats de carboni presenten una elevada variabilitat estereoquímica i els organismes s'aprofiten d'aquesta elevada variabilitat utilitzant oligosacàrids i polisacàrids per a una multitud de diferents funcions biològiques. A part de les típiques funcions estructurals i d'emmagatzematge energètic, tenen important rellevància en altres funcions molt més específiques, com per exemple en senyalització cel·lular. Aquesta funcionalitat desperta l'interès de multitud d'aplicacions biotecnològiques, que són el fruit d'un nombre creixent d'estudis en glicobiologia.L'enllaç glicosidic, en especial entre dues unitats de glucosa, és un dels enllaços més estables que es troben en biopolímers naturals. El temps de vida mitja de la hidròlisi espontània d'aquest enllaç en cel·lulosa i en midó és de l'ordre dels 5 milions d'anys. Les glicosil hidrolases son els enzims responsables de la hidròlisi enzimàtica d'aquests biopolímers. Aquests enzims aconsegueixen portar a terme la hidròlisi en escales de temps de l'ordre de 1000 cicles per segon. Donada aquesta elevada activitat, les glicosil hidrolases són considerades un dels catalitzadors biològics més eficients.La present tesi centra el seu principal interès en l'estudi de la formació del complex de Michaelis entre una glicosil hidrolasa de la família 16 (la 1,3-1,4-&#946;-glucanasa) i el seu corresponent substrat. Entendre el procés en el que el substrat s'uneix a l'enzim és un punt clau de cara a raonar les següents etapes de qualsevol mecanisme enzimàtic. Sovint aquest procés ja porta associats canvis conformacionals tant a nivell d'enzim com de substrat que contribueixen a la catàlisi, fent variar les diferències energètiques en el pas de reactius a productes. Per a les &#946;-glicosil hidrolases, existeixen diferents evidències experimentals que recolzen aquest fet. Així, s'observen estructures distorsionades a nivell de substrat respecte les que correspondrien a les estructures més estables en solució. De totes maneres, es desconeixen els factors que determinen que el substrat prefereixi unir-se a l'enzim en una conformació o una altra, i les implicacions mecanístiques que això té. La present tesi tracta de donar resposta a algunes d'aquestes qüestions emprant diferents tècniques de dinàmica molecular (clàssica i de primers principis) en combinació amb mètodes que permeten l'acceleració d'esdeveniments al llarg d'una simulació.La present tesi està estructurada en tres grans blocs: un bloc introductori (capítols I i II) on s'introdueix la família d'enzims a la qual pertany la 1,3-1,4-&#946;-glucanasa i s'estableixen els objectius generals del present estudi; al capítol II es resumeixen els fonaments de la metodologia computacional emprada. En un segon bloc (capítols III a VII) es descriuen les simulacions portades a terme per tal de donar resposta a les preguntes obertes plantejades al bloc introductori. Els resultats són analitzats i discutits en cinc capítols diferents: al capítol III s'analitza l'estructura del complex de Michaelis de la 1,3-1,4-&#946;-glucanasa amb un substrat tipus metilumbeliferil-tetrasacàrid; al capítol IV s'avalua i es descriu el mapa d'energia lliure conformacional de l'anell de &#946;-D-glucopiranosa, i s'analitzen les propietats estructurals i electròniques de cada conformació per tal de trobar una correlació amb les distorsions presents en diferents glucosidases; al capítol V s'avalua i es descriu el mapa d'energia lliure conformacional del substrat un cop unit a la 1,3-1,4-&#946;-glucanasa, i s'analitza l'efecte de diferents mutacions puntuals del substrat; al capítol VI es porta a terme una simulació del primer pas de la reacció enzimàtica de la 1,3-1,4-&#946;-glucanasa i s'analitza l'itinerari conformacional seguit pel substrat; al capítol VII es porta a terme una simulació del procés d'entrada del substrat a la cavitat enzimàtica i s'analitzen les reorganitzacions a nivell de substrat i de proteïna que tenen lloc al llarg del procés. Cada capítol conté una breu introducció específica de cada cas concret, informació sobre els detalls computacionals emprats, i la descripció i discussió dels resultats obtinguts. Finalment al darrer bloc (capítol VIII) es resumeixen els resultats més rellevants obtinguts en la present tesi i s'enumeren les principals conclusions. Addicionalment, s'han adjuntat tres annexes que complementen la informació dels diferents capítols. / Carbohydrates exhibit a high stereo chemical diversity. Living organisms take benefit of it by using oligosaccharides and polysaccharides for a large number of biological functions. In addition to the typical structural and energetic functions, carbohydrates play an important role in other more specific functions such as cellular signalling. This functionality is of special interest in different biotechnological applications as a result of an increasing number of studies in glycobiology.The glycosidic bond, specially the one between two glucose units, is one of the most stable bonds found in natural biopolymers. The half life time for the spontaneous hydrolysis of this bond in cellulose and starch is of the order of 5 million years. Glycosil hydrolases (GHs) are the enzymes responsible for the enzymatic hydrolysis of these biopolymers. GHs are able to hydrolyze the glycosidic bond in a time scale of the order of 1000 cycles per second. Due to this extremely high activity, GHs are considered some of the most efficient biological catalysts. This thesis is focused on the formation of the Michaelis complex in 1,3-1,4-&#946;-glucanase, a family 16 glycosil hydrolase. Understanding the substrate binding process is a key point to rationalize the subsequent steps of any enzymatic process. Usually, this process involves conformational changes of both the enzyme and the substrate. These changes contribute to the catalysis by changing the relative energies of reactants and products. Recent experiments on &#946;-glycosil hydrolases give evidence of a distorted structure of the substrate with respect to its structure in solution. However, the factors that dictate the preference of the substrate to be bound to the enzyme in one or another conformation remain unknown, as well as their mechanistic implications. This thesis tries to answer some of these questions using different techniques based on molecular dynamics (both classical and first principles) in combination with methods aimed to accelerate the simulation of rare events.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Katherine Arens. Vita. Includes bibliographical references.

This thesis summarizes the results of studies on two aspects of nucleic acids. Chemically modified antisense oligonucleotides (AONs) have been evaluated with regards to their suitability for mRNA targeting in an antisense approach (Paper I – III). The chemically modified nucleotidic units 2'-O-Me-T, 2'-O-MOE-T, oxetane-T, LNA-T, azetidine-T, aza-ENA-T, carbocyclic-ENA-T and carbocyclic-LNA-T were incorporated into 15-mer AONs and targeted against a 15-mer RNA chosen from the coding region of SV-40 large T antigen. The comparative study showed that a single modified nucleotide in the AON with North-East locked sugar (oxetane-T and azetidine-T) lowered the affinity for the complementary RNA whereas North locked sugars (LNA-T, aza-ENA-T, carbocyclic-ENA-T, and carbocyclic-LNA-T) significantly improved the affinity. A comparative RNase H digestion study showed that modifications of the same type (North-East type or North type) in different sequences gave rise to similar cleavage patterns. Determination of the Michaelis-Menten parameters by kinetic experiments showed that the modified AONs recruit RNase H resulting in enhanced turnover numbers (kcat) although with weaker enzyme-substrate binding (1/Km) compared to the unmodified AON. The modified AONs were also evaluated with regards to resistance towards snake venom phosphodiesterase and human serum to estimate their stability toward exonucleases. The aza-ENA-T and carbocyclic-ENA-T modified AONs showed improved stability compared to all other modified AONs. In general, the modified AONs with North type nucleotides (except LNA-T) were found to be superior to the North-East type as they showed improved target affinity, comparable RNase H recruitment capability and improved exonuclease stability. The second aspect studied in this thesis is based on physicochemical studies of short RNA molecules utilizing NMR based pH titration and alkaline hydrolysis reactions (Paper IV – V). The NMR based (1H and 31P) pH titration studies revealed the effect of guaninyl ion formation, propagated electrostatically through a single stranded chain in a sequence dependent manner. The non-identical electronic character of the internucleotidic phosphodiesters was further verified by alkaline hydrolysis experiments. The internucleotidic phosphodiesters, which were influenced by guaninyl ion formation, were hydrolyzed at a faster rate than those sequences where such guaninyl ion formation was prevented by replacing G with N1-Me-G.

In 2019, Adams, Ehlting, and Edwards developed a four-variable system of ordinary differential equations modelling phenylalanine metabolism in plants according to Michaelis-Menten kinetics. Analysis of the model suggested that when a series of reactions known as the Shikimate Ester Loop (SEL) is included, phenylalanine flux into primary metabolic pathways is prioritized over flux into secondary metabolic pathways when the availability of shikimate, a phenylalanine precursor, is low. Adams et al. called this mechanism of metabolic regulation the Precursor Shutoff Valve (PSV). Here, we attempt to simplify Adams and colleagues’ model by reducing the system to three variables and replacing the Michaelis-Menten terms with piecewise-defined approximations we call ramp functions. We examine equilibria and stability in this simplified model, and show that PSV-type regulation is still present in the version with the SEL. Then, we define a class of systems structurally similar to the simplified Adams model called biochemical ramp systems. We study the properties of the Jacobian matrices of these systems and then explore equilibria and stability in systems of n ≥ 2 variables. Finally, we make several suggestions regarding future work on biochemical ramp systems. / Graduate

In dieser Arbeit wurden zwei katalytische Modellreaktionen studiert. Zunächst die katalytische Reduktion von p-Nitrophenol (Nip) mit Natriumborhydrid (BH_4^-). Diese verläuft entlang der direkten Route: Dabei wird Nip über p-Hydroxylaminophenol (Hx) zum Produkt p-Aminophenol (Amp) reduziert. Ein kinetisches Modell wird vorgestellt, dass die Reaktion auf Basis des Langmuir-Hinshelwood (LH) Mechanismus beschreibt. Die Lösung der Gleichungen gibt die Nip Konzentration als Funktion der Zeit, welche direkt mit den experimentellen Daten verglichen werden kann. Werden als Katalysator auf sphärischen Polyeletrolytbürsten stabilisierte Gold Nanopartikel (SPB-Au) verwendet, zeigt sich eine gute Übereinstimmung und unterstreicht die Allgemeingültigkeit der direkten Route. Der zweite Teil beschäftigt sich mit der katalytischen Oxidation von 3,3’,5,5’-Tetramethylbenzidin (TMB) durch Wasserstoffperoxid (H_2O_2) an SPB-Pt Nanopartikeln. Dabei wurden die Katalyse mithilfe zweier Modelle analysiert: Michaelis-Menten (MM) und Langmuir-Hinshelwood (LH). Im MM Modell wird die Oxidation von TMB durch die Nanopartikel mit der Peroxidase katalysierten TMB Oxidation unter Annahme des Ping-Pong Mechanismus verglichen. Es wurde gezeigt, dass die häufig verwendete Analyse der initialen Reaktionsraten große Fehler verursacht und zu inkonsistenten Ergebnissen führt. Dies zeigt dass dieses Vorgehen zu Analyse der Oxidation von TMB nicht geeignet ist. Im LH Modell wird angenommen dass H_2O_2 und TMB im ersten Schritt auf der Oberfläche der Nanopartikel adsorbieren. Das LH Modell mit Produktinhibition ermöglicht hierbei eine zufriedenstellende Beschreibung der kinetischen Daten bis zu einem Umsatz von 40 %. Die gesamte Analyse zeigt, dass das Langmuir-Hinshelwood Modell die bessere Näherung zur Beschreibung der Kinetik der Nanopartikel katalysierten TMB Oxidation bietet / In this work, two catalytic model reactions were studied using different metallic nanoparticles in aqueous solution. One is the catalytic reduction of p-nitrophenol (Nip) by sodium borohydride (BH_4^-). The reaction proceeds in the following route: Nip is first reduced to p-hydroxylaminophenol (Hx) which is further reduced to the final product p-aminophenol (Amp). Here we present a full kinetic scheme according to Langmuir-Hinshelwood mechanism (LH). The solution of the kinetic equations gives the concentration of Nip as the function of time, which can be directly compared with the experimental data. Satisfactory agreement is found for reactions catalyzed by Au nanoparticles immobilized in spherical polyelectrolyte brushes (SPB-Au) verifying the validity of the reaction route. In the second part, we present a study on the catalytic oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) by hydrogen peroxide (H_2O_2) with SPB-Pt nanoparticles. The catalysis was analyzed by two different models: Michaelis-Menten (MM) and Langmuir-Hinshelwood (LH) model. In the MM model, the oxidation of TMB catalyzed by nanoparticles is inferred to the catalysis of peroxidase assuming the Ping-Pong mechanism. It is found that the frequently used analysis with the initial rates introduces large errors and leads to inconsistent results, which indicates that such approach is not suitable to analyze the oxidation of TMB catalyzed by nanoparticles. In the LH model, it is assumed that H_2O_2 and TMB adsorb on the surface of nanoparticles in the first step. The LH model with product inhibition gives satisfactory description of the kinetic data up to a conversion of 40%. The entire analysis demonstrates that the Langmuir-Hinshelwood model provides a superior approach to describe the kinetics of TMB oxidation catalyzed by nanoparticles.